Vector

ABSTRACT

The present invention provides a vector which can make nucleic acids to be an aggregate and locate a vector containing a cationic polymer to surround the aggregate so as to protect the nucleic acids from the enzyme. The vector comprises a polymer having branched chain(s). Preferably, 3, 4, or 6 branched chains are bonded to benzene ring. It is preferable that the number of the branched chains is higher. As the branched chain(s), a vinyl-series acrylic polymer is preferable. The vector is synthesized by reacting a dithiocarbamate compound with an acrylamide monomer for the branched chain(s).

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation Application of PCT/JP2004/005450 filed on Apr.16, 2004. The entire disclosure of the prior application is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a vector on gene transfectiontechnology.

BACKGROUND OF THE INVENTION

As the molecular genetic factors on human disorders became clearer,study for gene therapy has been increasingly emphasized recently. Genetherapy is intended to express DNA on targeted regions. The importantthings for gene therapy are how to deliver the DNA to the targetedregion, and how to effectively introduce the DNA into the targetedregion and express functionally the DNA on the region. As a vector forintroducing foreign DNA, many viruses including retrovirus, adenovirus,and herpesvirus, which are altered to transfer therapeutic gene, havebeen used for clinical trials on human gene therapy. However, the risksof infection and immune reaction still remain.

As a non-viral vector for transferring DNA into cells, for example,dioleoyloxypropyl trimethylammonium, which is a cationic lipid (Felgneret al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417, 1987), has beencommercialized as Lipofectin (registered trademark). As a syntheticpolymer for vector, polyethyleneimine has been commercialized as Exogen.

DNA-lipid complexes are described in Felgner et al., PNAS, 84 (1987)7413.

Generally, nucleic acids are not stable within a living body and aredecomposed by a certain type of enzyme.

SUMMARY OF THE INVENTION

The present invention provides a vector which can make nucleic acids tobe an aggregate and locate a vector containing a cationic polymer tosurround the aggregate so as to protect the nucleic acids from theenzyme.

The vector of the present invention comprises a cationic polymer havingbranched chain(s).

The vector of the present invention forms a complex with the aggregateof nucleic acids and can be administered into a living body.

As a device for insertion into living body, there are devices to bepercutaneously inserted into tissues near diseased parts and devices tobe placed within blood vessels such as a blood vessel catheter and astent graft. However, the device is not limited thereto.

Generally, nucleic acids are not stable in living body and aredecomposed by a certain type of enzyme. A nucleic-acid-containingcomplex using the vector of the present invention can make nucleic acidsto be an aggregate and locate a vector containing a cationic polymer tosurround the aggregate so as to protect the nucleic acids from theenzyme, thereby making at least nucleic acids inside the aggregate tooperate normally in the living body. In addition, thenucleic-acid-containing complex can be enclosed in a micro- ornano-order sized capsule or micelle and thus can be administered into ablood vessel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing measurement results of the particledistribution of a polyion complex.

FIG. 2 is a graph showing changes with time in particle size of thepolyion complex.

FIG. 3 is a graph showing results of transfection experiments.

FIG. 4 is a graph showing measurement results of the relation betweenthe cation/anion ratio and the gene expression efficiency.

FIG. 5 is a TEM photograph of a gene complex, which is stained withphosphotungstic acid.

FIG. 6 is a TEM photograph of a gene complex, which is stained withuranium acetate.

FIG. 7 is a graph showing the relation between the molecular weight of avector having a single branched chain and the gene expressionefficiency.

FIG. 8 is a graph showing the relation between the molecular weight of avector having three branched chains and the gene expression efficiency.

FIG. 9 is a graph showing the relation between the molecular weight of avector having four branched chains and the gene expression efficiency.

FIG. 10 is a graph showing the relation between the molecular weight ofa block polymer and the gene expression efficiency, wherein the blockpolymer is a vector having four branched chains and a molecular weightof 10,000 and is prepared by block polymerizing dimethylacrylamide onends of the branched chains; and

FIG. 11 is a graph showing the stability within the solution (changeswith time when the initial value of the gene expression efficiency isset to 100%) of a block polymer of the vector, a branched polymer, and aPEI.

PREFERRED EMBODIMENTS OF THE INVENTION

Cationic polymer employed in the present invention has branchedchain(s).

In an aspect of the present invention, the effect of vector is enhancedwithin a certain molecular weight range. The range depends on the numberof the branched chain(s).

In an aspect of the present invention, a vector has three polymer chainsand a molecular weight (number average molecular weight) of from 30,000to 60,000.

In an aspect of the present invention, a vector has four polymer chainsand a molecular weight of from 30,000 to 65,000.

In an aspect of the present invention, a vector has six polymer chainsand a molecular weight of from 10,000 to 50,000.

In an aspect of the present invention, a vector includes nonionic groupswhich are bonded to ends of the branched chains of the polymer.

In an aspect of the present invention, the vector is composed of apolymer which comprises a polyfunctional compound as its nucleus, aplurality of polymer chains as the branched chains which are bonded tothe nucleus, and nonionic groups which are bonded to the ends of thepolymer chains. The nonionic groups may be aromatic compounds, aliphaticcompounds, polyacrylic esters, or polymethacrylic esters.

In a preferred embodiment of the present invention, a plurality ofbranched chains are bonded to a polyfunctional compound, preferablybenzene ring, naphthalene, anthracene, or pyrene. In the case of benzenering, the number of bonds is from 2 to 6, especially 2, 3, 4, or 6. Thelarger the number of bonds, the more effective it is. In the case ofnaphthalene, the number of bonds can be up to 8. In the case ofanthracene or pyrene, the number of bonds can be up to 10. In addition,biphenyl (in this case, the number of bonds of branched chains can alsobe up to 10) in which a plurality of aromatic rings in noncoplanarrelation are bonded or a cycloparaffin hydrocarbon such as cyclohexanemay be employed as the nucleus.

As the nucleus to which the branched chains are bonded, benzene ring ispreferable. Specific examples are as follows. An example for threebranched chains is 2,4,6-tris (N,N-diethyldithiocarbamylmethyl)mesitylene which is obtained by addition reaction between 2,4,6-tris(bromomethyl) mesitylene and sodium N,N-diethyldithiocarbamate inethanol. An examples for four branched chains is 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl) benzene which is obtained by anaddition reaction between 1,2,4,5-tetrakis (bromomethyl) benzene andsodium N,N-diethyldithiocarbamate in ethanol. An example for sixbranched chains is hexakis (N,N-diethyldithiocarbamylmethyl) benzenewhich is obtained by an addition reaction between hexakis (bromomethyl)benzene and sodium N,N-diethyldithiocarbamate in ethanol.

As for the branched chain, homopolymer or copolymer of vinyl seriesmonomer is preferable. The length of each branched chain may be eitherthe same or different. The structure of the chain may be either linearstraight-chain structure or dendritic branched-chain structure. When thebranched chain is a homopolymer, specifically, a polymer of3-N,N-dimethylaminopropyl acrylamide, CH₂═CHCONHC₃H₆N(CH₃)₂, ispreferable.

By mixing the 3-N,N-dimethylaminopropyl acrylamide and theaforementioned each benzene derivative with an alcohol solution such asmethanol or a solution of low polarity solvent such as chloroform inconsideration of the solubility and subjecting the mixture tophotopolymerization reaction, a cationic polymer in which theaforementioned polymer of vinyl series monomer is bonded to the benzenering via —CH₂— derived from the aforementioned benzene derivative isproduced. The molecular weight of the thus produced vector is preferablyfrom 5,000 to 500,000, especially 5,000 to 100,000, particularly 10,000to 50,000. In addition, when the branched chain is a copolymer, it maybe either a random copolymer or a block copolymer. If it includes acationic functional monomer as the monomer, a copolymer of a nonionicmonomer or a hydrophobic monomer may be employed. As the nonionicmonomer, nonionic hydrophilic monomers such as N,N-dimethylacrylamide,N-vinylpyrrolidone, and PEG-(meth) acrylate are preferable. As thehydrophobic monomer, ethyl (meth) acrylate and styrene are preferable.The vector composed of thus produced cationic polymer surrounds thenucleic acids and functions as a nucleic-acid-containing complex,thereby inhibiting the deactivation and decomposition of the nucleicacids due to enzyme within a living body.

The polymer chain composing the branched chain may be a polymer chainwhich has a quaternary amine on its proximal end and a tertiary amine onits distal end. When quaternary amine is placed near the nucleus towhich the branched chain is bonded and tertiary amine is placed aroundthe quaternary amine, reduction of cytotoxicity and high efficiency ofgene transfection could be expected. Quaternary amine is cation which isan essential ingredient of a vector. However, quaternary amine hascytotoxicity as it is used as a fungicide or an antibacterial coating.Therefore, placing tertiary amine around the quaternary amine would beof great advantage in cytotoxicity. As a matter of course, besides thecombination of quaternary and tertiary, combinations of such asquaternary and primary, quaternary and secondary, tertiary and primary,tertiary and secondary may be possible.

Moreover, the cationic polymer within the vector of the presentinvention may have such a nature that the positive charge thereof isdeactivated with time. Since the positive charge within the vector isdeactivated as mentioned above, the function of emission of the nucleicacids from the nucleic acid aggregate which is surrounded by the vectorcan be given. The conditions may be set by a person skilled in the artin such a manner that the emission of nucleic acids will be conductedafter the vector permeates the cell membrane. The cationic polymerhaving such function may be a polymer carrying a compound having4,4′-benzylidenebis (N,N-dimethylaniline) skeleton on its branchedchains of a long-chain form. The compounds having 4,4′-benzylidenebis(N,N-dimethylaniline) skeleton may be leucomalachite green. The cationicpolymer carrying the aforementioned compounds is converted to cation bylight irradiation, and has a nature that the positive charge thereof isdeactivated with time.

To combine the vector composed of cationic polymer and the nucleicacids, the nucleic acids are added to and mixed with dispersion liquidin which the concentration of the vector is from about 1 to about 1,000μg/mL at room temperature. It is preferable to add the cationic polymerto the nucleic acids in an excessive amount so as to make anucleic-acid-containing complex in which the cationic polymer iscombined with the nucleic acid in the saturated state of the cationicpolymer.

As the nucleic acid, polynucleotide such as deoxyribonucleic acid (DNA)and ribonucleic acid (RNA) are preferable. Among these, DNA isespecially preferable. The nucleic acid may be ribonucleoprotein.

The nucleic acid may be herpes simple virus thymidine kinase gene(HSV1-TK gene), p53 tumor suppressor gene, and BRCA1 tumor suppressorgene, cytokine gene such as TNF-α gene, IL-2 gene, IL-4 gene,HLA-B7/IL-2 gene, HLA-B7/B2M gene, IL-7 gene, GM-CSF gene, IFN-γ geneand IL-12 gene, or cancer antigen peptide gene such as MART-1 andMAGE-1. These are available for cancer therapy.

Moreover, cytokine gene such as VEGF gene, HGF gene, and FGF gene, c-mycantisense, c-myb antisense, cdc2 kinase antisense, PCNA antisense, E2Fdecoy, and p21 (sdi-1) gene are available for blood vessel diseasetreatment. These genes are well known to those skilled in the art.

The particle size of the nucleic-acid-containing complex is preferablyfrom about 50 to about 400 nm. If it is smaller than this range, thereis possibility that the effect of the enzyme affects the nucleic acidsin the nucleic-acid-containing complex and there is also possibilitythat the nucleic-acid-containing complex is filtered out at the kidney.Moreover, if it is larger than this range, there is possibility that thenucleic-acid-containing complex has difficulty in introduction intocells.

Nucleic acid is used in a form that it can exhibit its function withincells by the introduction into the cells. For instance, in the case ofDNA, nucleic acid is used in the form of plasmid in which the DNA istranscribed within cells where the nucleic acid is introduced and isarranged such that the DNA can exhibit its function through productionof polypeptide to be encoded. Preferably, promoter region, initiationcodon, DNA which codes protein having desired function, terminationcodon, and termination region are seriated.

If required, two or more kinds of nucleic acids may be included in asingle plasmid.

According to the present invention, “cells” preferable for introductionof nucleic acid are cells which allow the nucleic acid to exhibit itsfunction and are selected diversely according to the nucleic acid to beused (i.e., the function thereof). Examples of such cells includecardiac muscle cell, smooth muscle cell, fibrocyte, skeletal musclecell, vascular endothelial cell, bone marrow cell, bone forming cell,hematocyte stem cell, blood cell, tumor cell, hemopoietic stem cell,peripheral stem cell, SP cell, ES cell, B cell, T cell, NK cell.Moreover, the examples also include gastrointestinal tract epithelialcells and renal tubular epithelial cells such as monocyte, dendriticcell, macrophage, histiocyte, Kuppfer cell, osteoclast, synovial A cell,microgliocyte, Langerhans cell, epithelioid cell, multinucleate giantcell.

Nucleic-acid-containing complex using the vector of the presentinvention can be administered in any given manner.

As for the administration, intravenous or intraarterial infusion isespecially preferable. However, the nucleic-acid containing complex maybe administered into muscle, into adipose tissues, under the skin,intradermally, into lymphatic vessels, into lymph nodes, into bodycavities (pericardial cavity, pleural cavity, abdominal cavity,cerebrospinal cavity, and the like), and into bone marrow. Besidesthese, the nucleic-acid containing complex may be administered directlyinto diseased tissues, for example, spraying alveolus with thenucleic-acid containing complex by using a tracheal endoscope.

Drugs of which active ingredient is the nucleic-acid-containing complexcan be further mixed with carrier (osmotic adjuster, stabilizer,preservative, solubilizer, pH adjuster, thickener, and the like)acceptable in pharmaceutical preparations if necessary. These carriersmay be of heretofore known type. Moreover, an indirect manner which iswell known to those skilled in the art and in which thenucleic-acid-containing complex is used outside the body first and thenadministered into a living body may be employed. For instance, theindirect manner comprises letting the nucleic-acid-containing complexaffect cells taken from the living body such as lymph cells having hightumor cytotoxicity to give drug sensitive to the cells and, after that,administrating them into the living body after incubation. In this case,it is possible to injure the tumor cells with the lymph cells incubated,and kill the administered lymph cells by drugs if any side effect isconfirmed.

Moreover, drugs of which active ingredient is thenucleic-acid-containing complex include drugs containing two or morekinds of nucleic-acid-containing complexes of which nucleic acids aredifferent from each other. Drugs having several therapeutic purposes asmentioned above are especially helpful in the field of the diversifyinggene therapys.

As for the administration quantity of such drug, the quantity to animal,especially the quantity to human, depends on various factors such as thetargeted nucleic acids, the administration manner, and the particularpart to be treated. However, the administration quantity should besufficient enough to cause remedial response.

The nucleic-acid-containing complex therein is preferably applied forgene therapy. The applicable diseases depend on the kind of the nucleicacids contained in the complex. Specific examples include peripheralarterial disease, coronary artery disease, and diseases which are causedat circulatory organ region after arterial dilatation such asrestenosis. The examples also include cancers (malignant melanoma, braintumor, metastasizing malignancy, breast cancer, and the like), infectiondiseases (such as HIV), and monogenic disorders (cystic fibrosis,chronic granulomatous disease, alpha-1-antitrypsin deficiency, Gaucher'sdisease, and the like).

EXAMPLES Example 1 Synthesis of Single Branched Chain Type Vector

First, benzyl N,N-diethyldithiocarbamate was synthesized according tothe following reaction formula (Formula 1).

benzyl N,N-diethyldithiocarbamate

More specifically, 10 ml of ethanol solution containing benzyl chloride(4.8 g, 38 mmol, Mw. 126.58) was added dropwise into 50 ml of ethanolsolution containing sodium N,N-diethyldithiocarbamate trihydrate (10.3g, 46 mmol, Mw. 225.31) in nitrogen atmosphere at 0° C. After agitatingthe reaction solution for 23 hours at room temperature, 150 ml of waterwas added and extracted by diethylether (200 ml×3 times). The organiclayer was washed with water (100 ml×3 times) and was dried by sodiumsulfate. Solvent was distilled away under reduced pressure using anevaporator so as to obtain benzyl N,N-diethyldithiocarbamate (colorlessliquid). Yield point was 17.6 g (yield: 93%), ¹H-NMR: δ7.407 ppm to7.271 ppm (m, 5H, Ar—H), δ4.540 ppm (s, 2H, Ar—CH₂S), δ4.082 ppm to4.012 ppm (q, 2H, —N—CH₂—), δ3.763 ppm to 3.692 ppm (q, 2H, —N—CH₂—),δ1.311 to 1.252 ppm (m, 6H, —CH₂—CH₃).

Then, using the benzyl N,N-diethyldithiocarbamate thus obtained,dimethylaminopropyl acrylamide was polymerized according to thefollowing reaction formula (Formula 2).

More specifically, a mixture of dimethylaminopropyl acrylamide (3.9 g,24.96 mmol, Mw. 156.23) and benzyl N,N-diethyldithiocarbamate (23.94 mg,0.1 mmol, Mw. 239.41) was diluted with methanol so as to preparesolution of 20 mL in total. The solution thus prepared was agitatedwhile nitrogen gas was blown in, and was irradiated with ultravioletlight (light intensity 1 mW/cm²). After 30 minutes of ultravioletirradiation, polymer solution was concentrated using an evaporator andwas added dropwise to a large quantity of diethylether so as toprecipitate high-molecular-weight polymer. Supernatant liquid wasremoved by decantation and the high-molecular-weight polymer wasdissolved in water and was freeze-dried. Molecular weight of thehigh-molecular-weight polymer was measured after freeze-dried, and wasapproximately 18,000, ¹H-NMR: δ7.8 ppm to 7.4 ppm (br, 1H, —NH), δ3.43ppm to 3.0 ppm (br, 2H, —NH—CH₂—CH₂—), δ2.4 ppm to 2.2 ppm (br, 2H,—CH₂—CH₂—NR₂), δ2.2 ppm to 2.1 ppm (br, 6H, —N—CH₃), δ1.8 ppm to 1.5 ppm(br, 2H, —CH₂—CH₂—CH₂—).

Example 2 Synthesis of 3 Branched Chain Type Vector

Next, 2,4,6-tris (N,N-diethyldithiocarbamylmethyl) mesitylene wassynthesized by reacting sodium N,N-diethyldithiocarbamate with2,4,6-tris (bromomethyl) mesitylene according to the following reactionformula (Formula 3).

2,4,6-tris (N,N-diethyldithiocarbamylmethyl) mesitylene

More specifically, ethanol (100 ml) and sodiumN,N-diethyldithiocarbamate trihydrate (5.43 g, 24.12 mmol, Mw. 225.31)were added to 2,4,6-tris (bromomethyl) mesitylene (2 g, 4.02 mmol, Mw.398.98) and were agitated at room temperature. After 24 hours from thestart of the agitation, the mixture was filtered and the precipitate wascollected. The collected precipitate was dissolved in chloroform, andwas washed with water using a separatory funnel. Thus formed chloroformlayer was concentrated using an evaporator and vacuum-dried using adesiccator, thereby obtaining 2,4,6-tris(N,N-diethyldithiocarbamylmethyl) mesitylene (white solid). Yield pointwas 3.03 g (yield: 98.4%), ¹H-NMR: δ4.447 ppm (s, 6H, Ar—CH₂S), δ4.086ppm to 4.015 ppm (q, 6H, —N—CH₂—), δ3.746 ppm to 3.676 ppm (q, 6H,—N—CH₂—), δ2.421 ppm (s, 9H, Ar—CH₃), δ1.324 ppm to 1.222 ppm (m, 18H,—CH₂—CH₃).

To 2,4,6-tris (N,N-diethyldithiocarbamylmethyl) mesitylene thusobtained, dimethylaminopropyl acrylamide was polymerized according tothe following reaction formula (Formula 4), thereby synthesizing a threebranched chain type vector.

More specifically, 2,4,6-tris (N,N-diethyldithiocarbamylmethyl)mesitylene (20.14 mg, 1.67 mmol, Mw. 604.08) was dissolved in chloroform(300 μl) and was added to methylaminopropylacrylamide (1.6224 g, 10.38mmol, Mw. 156.23). The mixture was diluted with methanol to preparesolution of 20 ml in total. The solution thus prepared was agitatedwhile nitrogen gas was blown in, and was irradiated with ultravioletlight (light intensity 1 mW/cm²). After 30 minutes of ultravioletirradiation, polymer solution was concentrated using an evaporator andwas added dropwise to a large quantity of diethylether to precipitatethe high-molecular-weight polymer. Supernatant liquid was removed bydecantation and high-molecular-weight polymer was dissolved in water andwas freeze-dried. Molecular weight of the obtained high-molecular-weightpolymer was measured using a GPC after freeze-dried and wasapproximately 18,000. In addition, ¹H-NMR: δ7.7 ppm to 7.4 ppm (br, 1H,—NH), δ3.4 ppm to 3.0 ppm (br, 2H, —NH—CH₂—CH₂—), δ2.4 ppm to 2.3 ppm(br, 2H, —CH₂—CH₂—NR₂), δ2.3 ppm to 2.1 ppm (br, 6H, —N—CH₃), δ1.8 ppmto 1.5 ppm (br, 2H, —CH₂—CH₂—CH₂—).

Example 3 Synthesis of Four Branched Chain Type Vector

First, 1,2,4,5-tetrakis (N,N-dithiocarbamylmethyl) benzene wassynthesized according to the following reaction formula (Formula 5).

1,2,4,5-tetrakis (N,N-dithiocarbamylmethyl)

More specifically, ethanol (100 mL) and sodiumN,N-diethyldithiocarbamate trihydrate (4 g, 17.76 mmol, Mw. 225.31) wereadded to 1,2,4,5-tetrakis (bromomethyl) benzene (1 g, 2.22 mmol, Mw.449.83) and were agitated at room temperature. After 48 hours from thestart of agitation, the mixture was filtered and the precipitate wascollected. The collected precipitate was dissolved in chloroform, andwas washed with water using a separatory funnel. The chloroform layerwas concentrated using an evaporator and was vacuum-dried using adesiccator, thereby obtaining 1,2,4,5-tetrakis(N,N-dithiocarbamylmethyl) benzene (white solid). Yield point was 1.48 g(yield: 91.4%). Measurement of ¹H-NMR resulted in δ7.487 ppm (s, 2H,Ar—H), δ4.573 ppm (s, 8H, Ar—CH₂S), δ4.065 ppm to 3.994 ppm (q, 8H,—N—CH₂—), δ3.765 ppm to 3.687 ppm (q, 8H, —N—CH₂—), δ1.304 ppm to 1.256ppm (t, 24H, —CH₂—CH₃).

To 1,2,4,5-tetrakis (N,N-diethyldithiocarbamylmethyl) benzene thusobtained, dimethylaminopropyl acrylamide was polymerized according tothe following reaction formula (Formula 6), thereby synthesizing a fourbranched chain type vector.

More specifically, 1,2,4,5-tetrakis (N,N-diethyldithiocarbamylmethyl)benzene (18.08 mg, 1.25 mmol, Mw. 723.30) was dissolved in chloroform(300 μl) and was added to dimethylaminopropyl acrylamide (1.326 g, 8.49mmol, Mw. 156.23). The mixture was diluted with methanol so as toprepare solution of 20 ml in total. The solution thus prepared wasagitated while nitrogen gas was blown in, and was irradiated withultraviolet light (light intensity 1 mW/cm²). After 30 minutes ofultraviolet irradiation, polymer solution was concentrated using anevaporator and was added dropwise to a large quantity of diethylether toprecipitate the high-molecular-weight polymer. Supernatant liquid wasremoved by decantation and high-molecular-weight polymer was dissolvedin water and was freeze-dried. Molecular weight was measured using a GPCafter freeze-dried, and was approximately 18,000. In addition, ¹H-NMR:δ7.8 to 7.4 ppm (br, 1H, —NH), δ3.4 ppm to 3.0 ppm (br, 2H,—NH—CH₂—CH₂—), δ2.4 ppm to 2.2 ppm (br, 2H, —CH₂—CH₂—NR₂), δ2.2 ppm to2.1 ppm (br, 6H, —N—CH₃), δ1.8 ppm to 1.5 ppm (br, 2H, —CH₂—CH₂—CH₂—).

Example 4 Synthesis of Six Branched Chain Type Vector

First, hexakis (N,N-diethyldithiocarbamylmethyl) benzene was synthesizedaccording to the following reaction formula (Formula 7).

hexakis (N,N-diethyldithiocarbamylmethyl) benzene

More specifically, ethanol (200 ml) and sodiumN,N-diethyldithiocarbamate trihydrate (6.36 g, 28.23 mmol, Mw. 225.31)were added to hexakis (bromomethyl) benzene (1 g, 1.57 mmol, Mw. 635.68)and were agitated at room temperature. After 4 days from the start ofagitation, the mixture was filtered and the precipitate was collected.The collected precipitate was dissolved in chloroform, and was washedwith water using a separatory funnel. The chloroform layer wasconcentrated using an evaporator and was vacuum-dried using adesiccator, thereby obtaining hexakis (N,N-diethyldithiocarbamylmethyl)benzene (white solid). Yield point was 1.48 g (yield: 90.2%). ¹H-NMR:δ4.565 ppm (s, 12H, Ar—CH₂S), δ4.012 to 3.988 ppm (q, 12H, —N—CH₂—),δ3.731 ppm to 3.708 ppm (q, 12H, —N—CH₂—), δ1.307 ppm to 1.261 ppm (m,36H, —CH₂—CH₃).

Then, dimethylaminopropyl acrylamide was polymerized to the hexakis(N,N-diethyldithiocarbamylmethyl) benzene thus obtained according to thefollowing reaction formula (Formula 8), thereby synthesizing a sixbranched chain type vector.

More specifically, hexakis (N,N-diethyldithiocarbamylmethyl) benzene(17.43 mg, 0.83 mmol, Mw. 1045) was dissolved in a small amount ofchloroform and was added to dimethylaminopropyl acrylamide (3.9 g, 24.96mol, Mw. 156.23). The mixture was diluted with chloroform so as toprepare solution of 20 ml in total. The solution thus prepared wasagitated while nitrogen gas was blown in, and was irradiated withultraviolet light (light intensity 1 mW/cm²). After 30 minutes ofultraviolet irradiation, polymer solution was concentrated using anevaporator, and was added dropwise to a large quantity of diethyletherto precipitate high-molecular-weight polymer. After supernatant liquidwas removed by decantation, the high-molecular-weight polymer wasdissolved in water and was freeze-dried. Molecular weight of thehigh-molecular-weight polymer thus obtained was measured using a GPCafter freeze-dried and was approximately 18,000. In addition, ¹H-NMR:δ7.8 ppm to 7.4 ppm (br, 1H, —NH), δ3.43 to 3.0 ppm (br, 2H,—NH—CH₂—CH₂—), δ2.4 ppm to 2.2 ppm (br, 2H, —CH₂—CH₂—NR₂), δ2.2 ppm to2.1 ppm (br, 6H, —N—CH₃), δ1.8 ppm to 1.5 ppm (br, 2H, —CH₂—CH₂—CH₂—).

(Formation of Nucleic-Acid-Containing Complex)

Each vector (2.36 mg, M.w. 18,000) was dissolved in tris-HCl-buffer (2ml). 50 μL of solution thus obtained was taken and was diluted by addingtris-HCl-buffer so as to prepare vector solution of 500 μl in total.Separately, 2× buffer solution (450 μl) was added to pGL3 controlplasmid so as to prepare DNA solution of 540 μl in total. The vectorsolution (67 μl) was added to the DNA solution (100 μl) (the ratio ofthe positive charge quantity of cationic high-molecular-weight polymerto the negative charge quantity of DNA was 1:1) and was left at rest for24 hours at 37° C., thereby forming a nucleic-acid-containing complex.Particle size of polyion complex was measured by dynamic lightscattering measurement and the result is shown in FIG. 1. In addition,changes of the average particle size with time are shown in FIG. 2.

As shown in FIGS. 1 and 2, each cationic high-molecular-weight polymerformed a polyion complex immediately after mixed with the DNA. Accordingto the result of cumulant analysis, it was found that nano-sizedparticles of approximately 250 nm were obtained and were still stableafter 24 hours.

Polyion complex solution was added at a rate of 25 μl per well to COS-1cells which has been incubated for a day on a 24 well-plate. The cellswere incubated in a 5% CO₂ incubator. After 3 hours of incubation, themedium was removed and the cells were washed with PBS. After that, DMEMwas added at a rate of 1 ml per well to the cells and the cells wereincubated within the 5% CO₂ incubator again. After two days ofincubation, the medium was removed and the cells were washed with PBS.After that, Luciferase cell culture lysis 5× Reagent was added at a rateof 200 μl per well and was left at rest. After 30 minutes, the cellswere put into an Eppendorf tube and were centrifuged (4° C., 15,000 rpm,1 min.). After centrifugation, each 4 μl of supernatant was collectedonto a microplate and luciferase activity was measured using aluminometer. Moreover, 5 μL of supernatant after centrifugation wascollected, and protein concentration determination was operated.

Transfection experiments were conducted using high-molecular-weightpolymer vectors having different numbers of branched chains. When C/Aratio was 10, the gene expression efficiency increased significantly asthe number of the branched chains of the high-molecular-weight polymerincreased as shown in FIG. 3. Effect on gene expression efficiencyaccording to variation of the C/A ratio (cation/anion ratio) was alsoexamined using a 6-branched-chain-type cationic high-molecular-weightpolymer which exhibited the highest gene expression efficiency in theabove experiments. As shown in FIG. 4, the highest gene expressionefficiency was obtained when C/A=40.

As mentioned above, the present invention provides a vector which canmake nucleic acids to be an aggregate and locate a vector containing acationic polymer to surround the aggregate so as to protect the nucleicacids from the enzyme.

FIGS. 5 and 6 are TEM photographs of gene complexes, each of which is amixture of PGL3 plasmid and the aforementioned 6-branched-chain-typevector, wherein FIG. 5 shows a gene complex which is stained withphosphotungstic acid and FIG. 6 shows a gene complex which is stainedwith uranium acetate.

FIG. 7 is a graph showing the relation between the molecular weight of avector having a single branched chain and the gene expressionefficiency.

FIG. 8 is a graph showing the relation between the molecular weight of avector having three branched chains and the gene expression efficiency.

FIG. 9 is a graph showing the relation between the molecular weight of avector having four branched chains and the gene expression efficiency.

FIG. 10 is a graph showing the relation between the molecular weight ofa block polymer and the gene expression efficiency, wherein the blockpolymer is a vector having four branched chains and a molecular weightof 10,000 and is prepared by block polymerizing dimethylacrylamide onends of the branched chains, and FIG. 11 is a graph showing thestability within the solution (changes with time when the initial valueof the gene expression efficiency is set to 100%) of a block polymer ofthe vector, a branched polymer, and a PEI.

1. A nucleic-acid-containing complex comprising a nucleic acid and avector, wherein the vector is represented by a Formula selected from thegroup consisting of Formula (I) and Formula (II):

wherein R₁-R₆ each represent polymer chains, wherein R₁-R₆ may each bethe same or different,

wherein R₇-R₁₀ each represent polymer chains, wherein R₇-R₁₀ may each bethe same or different, wherein the polymer chains represented by R₁-R₁₀are polymers consisting of dimethylaminopropyl acrylamide monomers, andwherein said nucleic-acid-containing complex has a particle size ofabout 50 to about 400 nm.
 2. The nucleic-acid-containing complex ofclaim 1, wherein the vector is of Formula II and has a molecular weightof from 30,000 to 65,000.
 3. The nucleic-acid-containing complex ofclaim 1, wherein the vector is of Formula I and has a molecular weightof from 10,000 to 50,000.
 4. A nucleic-acid-containing complex asclaimed in claim 1, wherein said vector is represented by Formula (I).5. The nucleic-acid-containing complex of claim 1, wherein the molecularweight of the vector is from 5,000 to 500,000.
 6. Thenucleic-acid-containing complex of claim 1, wherein the molecular weightof the vector is from 5,000 to 50,000.
 7. The nucleic-acid-containingcomplex as claimed in claim 1, wherein said nucleic-acid-containingcomplex has a particle size of approximately 250 nm, wherein acation/anion ratio of said nucleic-acid-containing complex is 20 to 160,and wherein the vector is represented by Formula (I) and has a molecularweight of approximately 18,000.